Not less than 60,000 bovine embryos are distributed per year in Japan. The demand for bovine embryos is increasing year by year in North America, South America, Europe and Asia. Thus, researches on cryopreservation of bovine embryos are being carried out worldwide, and development of a number of slow freezing methods and rapid freezing methods has realized relatively stable conception rates. However, the conception rate which can be achieved at the current technological level is only about 50%, and no significant improvement in the conception rate has been made during the last 20 or more years. This might indicate that cryopreservation of mammalian embryos has reached a certain technological limit.
Embryos resistant to damage caused by freezing/thawing are limited to high-quality embryos. Production of a plurality of embryos by superovulation treatment often results in obtaining only low-quality embryos due to various factors (for example, bad physical condition of the donor cattle or low vitality of the donated sperm). Nevertheless, these low-quality embryos can also be implanted into the uteri of recipient cattle to produce calves if the embryos have not undergone freezing treatment.
If there is any method available that allows survival of bovine embryos collected from donor cattle in the non-frozen state for a short period while suppressing their growth, embryos that have not suffered from injury by freezing/thawing can be very efficiently used for production of calves by utilization of transportation means such as home delivery services. Moreover, in cases where there is a time lag of several days between the estrous cycles of the donor cattle and the recipient cattle, the timing of implantation can be adjusted according to the estrous cycle of the recipient cattle. Furthermore, development of such a method for preserving embryos will eliminate the necessity of liquid nitrogen or an expensive refrigeration equipment such as a deep freezer. That is, preservation of embryos in a household refrigerator will become possible.
It has been reported that the limit of the period of non-freezing preservation is 3 days in the case of bovine embryos (Non-patent Document 1), and 4 days in the case of ovine embryos (Non-patent Document 2). A thermal hysteresis protein (Nfe8) and thermal hysteresis-like proteins Nfe11 and Nfe6, which are found in a fish species Zoarces elongatus Kner, have been reported to exert a cell life prolonging function under low-temperature conditions (Patent Document 1), and their practical use as cell life prolonging agents has been expected.
The animal species which have been studied for non-freezing low-temperature preservation include various species such as mouse, pig, sheep, cattle, and rare species. The properties of embryos are largely different among the animal species. Thus, components effective for embryo preservation, and mixing ratios and effective concentration ranges of these components, are also largely different among the species, and therefore components for the preservative solution need to be carefully examined for each animal species. The method of preservation of bovine embryos at non-freezing low temperature has long been studied from the viewpoints of improvement of the conception rate, estrus synchronization between the donor cattle and the recipient cattle, and utilization of low-quality embryos. However, no effective preservation method has been developed so far.
Under such circumstances, development of a preservative solution for mammalian embryos which not only enables more effective protection or preservation of mammalian embryos at non-freezing low temperature to prolong their life, but also achieves maintenance of hatching ability and a high conception rate, has been demanded. Similarly, development of a method for preserving mammalian embryos which not only enables their preservation for the number of days required for transportation, but also realizes their preservation and transportation using small general-purpose equipment or a container that consumes less energy, has been strongly demanded.